Field of the Invention
This invention relates to the construction of a recombinant Classical Swine Fever Virus (CSFV) live attenuated candidate strain vaccine, FPi.c. The FPi.c virus contains mutations in three amino acid residues within the fusion peptide (FP) region of CSFV E2 comprising amino acid residues 869-878 resulting in the mutations: W871T, W875D, and V878T.
Description of the Relevant Art
Classical swine fever (CSF) is a highly contagious disease of swine caused by CSF virus (CSFV), a small enveloped virus with a positive-sense, single-strand RNA genome. CSFV is classified as a member of the pestivirus genus within the Flaviviridae family along with other viruses of economic importance, bovine viral diarrhea virus (BVDV) and border disease virus (BDV) (Becher et al. 2003. Virology 311: 96-104). The approximately 12.5-kb CSFV genome contains a single open reading frame that encodes a polyprotein composed of 3,898 amino acids that ultimately yields up to 12 final cleavage products (NH2-Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH) through co- and post-translational processing of the polyprotein by cellular and viral proteases (Rice, C. M. 1996. In: Fundamental Virology, 3rd edition, Fields and Howley, eds., Lippincott Raven, Philadelphia, pp. 931-959).
Structural components of the virion include the capsid (C) protein and glycoproteins: Erns, E1 and E2. Erns, a secreted protein that demonstrates RNAse activity and is loosely associated with the viral envelope (Thiel et al. 1991. J. Virol. 65: 4705-4712; Weiland et al. 1990. J. Virol. 64: 3563-3569; Weiland et al. 1999. J. Gen. Virol. 80: 1157-1165) does not have a hydrophobic transmembrane anchor domain. Erns does, however, possess a C-terminal charged amphipathic segment that can mediate translocation of Erns across bilayer membranes (Langedijk, J. P. 2002. J. Biol. Chem. 277:5308-5314). E1 and E2 are transmembrane proteins with an N-terminal ectodomain and a C-terminal hydrophobic anchor (Thiel et al. 1991, supra). E2 is considered essential for CSFV replication, as virus mutants containing partial or complete deletions of the E2 gene are nonviable (van Gennip et al. 2002. Vaccine 20:1544-1556). E2 has been implicated, along with Erns (Hulst and Moorman. 1997. J. Gen. Virol. 78 (Pt 11): 2779-2787) and E1 (Wang et al. 2004. Virol. 330:332-341), in viral adsorption to host cells (Liang et al. 2003. J. Gen. Virol. 84:1269-1274; Van Gennip et al. 2000. Vaccine 19:447-459). Modifications introduced into this glycoprotein appear to have an important effect on CSFV virulence (Risatti et al. 2005. J. Virol. 79: 3787-3796; Risatti et al, 2006. Virology 355: 94-101; Risatti et al. 2007. J. Virol. 81: 924-933.; Van Gennip et al. 2004. J. Virol. 78: 8812-8823).
Using proteomic computational analysis, E2 has been characterized as a truncated class II fusion protein (Garry and Dash. 2003. Virol. 307:255-265). Although the overall structures of class I and II fusion proteins are distinct, they may share structural/functional characteristics in the parts of the molecules that interact with and disrupt bilayer membranes. It is well established that class I fusion proteins have a fusion peptide at the amino terminus of the molecule, or close to it, that is critical for fusion (Gallaher, W. R. 1987. Cell 50:327-328; Gallaher, W. R. 1996. Cell 85:477-478; Gallaher et al. 1989. AIDS Res. Human Retroviruses 5:431-440; Gallaher et al. 2001. BMC Microbiol. 1:1). Class II fusion proteins have an internal FP that is located after secondary structural folding at distal locations from the transmembrane anchor (Kuhn et al. 2002. Cell 108:717-725; Lescar et al. 2001. Cell 105:137-148; Rey et al. 1995. Nature 375:291-298)
Strategies for controlling disease in the event of a CSFV outbreak include the production of rationally designed live attenuated vaccine CSFV strains. Here, we report the effects of modifying a region within the CSFV structural glycoprotein E2, a region which contributes to the interaction between E2 and the host cell membrane.